High-yield expression of heterologous proteins in plants can be achieved using viral vectors. Viral vector systems were predominantly developed for transient expression followed by infection (Donson at al., 1991, Proc Natl Acad Sci USA, 88:7204-7208; Chapman, Kavanagh & Baulcombe, 1992, Plant J., 2:549-557) or transfection (Marillonnet et al., 2005, Nat Biotechnol., 23:718-723; Santi et al., 2006, Proc Natl Acad Sci USA. 103:861-866; WO2005/049839) of a plant host. The best-established and commercially viable systems are based on plus-sense single-stranded RNA viruses, preferably on Tobacco Mosaic Virus (TMV)-derived vectors (Kumagai at al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Mallory et al., 2002, Nature Biotechnol. 20, 622-625; U.S. Pat. No. 5,316,931; U.S. Pat. No. 5,589,367; U.S. Pat. No. 5,866,785; U.S. Pat. No. 5,977,438; WO02088369; WO02097080; WO0229068; U.S. Pat. No. 5,491,076).
Another group of RNA virus-based vectors derived from potexvirus PVX (potato virus X) can also provide for reasonably high yield of recombinant proteins, albeit noticeably lower yield than TMV-derived vectors (Chapman, Kavanagh & Baulcombe, 1992, Plant J., 2:549-557; Baulcombe, Chapman & Santa Cruz, 1995, Plant J., 7:1045-1053; Zhou et al., 2006, Appl. Microbiol. Biotechnol., April 13, epub ahead of print; Zelada at al., 2006, Tuberculosis, 86:263-267). Obviously, such system needs further improvement in order to increase the yield of recombinant protein of interest.
In the first generation of systemic viral vectors, a large proportion of plant resources was wasted for the production of viral coat protein that is necessary for systemic movement of a viral replicon. For TMV-derived vectors this problem was solved by removing the coat protein gene and by using agro-infiltration for efficient systemic delivery of replicons, thus significantly boosting the yield of recombinant proteins of interest (WO2005/049839; Marillonnet et al., (2005), Nat. Biotechnol., 23:718-723). However, unlike TMV-derived replicons, potexvirus-derived replicons require viral coat protein not only for systemic, but also for short distance (cell-to-cell) movement. Therefore, the coat protein gene of potexvirus-derived viral vectors cannot be removed without a severe loss of protein expression efficiency. Further, engineering of plant host providing viral coat protein in trans is rarely a good solution because of gene silencing. Also, transgenic plants expressing coat protein might exhibit coat protein-mediated resistance to challenges by plant viruses (Beachy, R N., 1999, Philos. Trans. R. Soc. Lend B Biol. Sci., 354:659-664; Wisniewski et al., 1990, Plant Cell, 2:559-567). Another similar phenomenon called heterologous CP-mediated resistance can be a problem, for example when a transgenic plant expressing PVX CP reduces cell-to-cell spread of TMV RNA (Bazzini et al., 2006, J. Gen. Virol., 87:1005-1012). In addition, expression of PVX coat protein in transgenic tobacco plants rescues movement-deficient PVX, but compromises the efficiency of cell-to-cell movement and viral replication (Spillane et al., 1997, Virology, 236:76-84). This could be an issue when the use of two viral vectors (e.g. TMV- and PVX-based vectors) in the same plant cell is required for expression of hetero-oligomeric proteins (e.g. for the co-expression of the heavy and light chains of a monoclonal antibody) (WO2006/079546; Giritch et al., 2006, Proc. Natl. Acad. Sci. USA, in press).